The purpose of the X-ray crystallography core facility is to provide equipment, training, assistance, and technological innovations for determining three-dimensional structures of protein and other macromolecules. Services provided by the facility include crystallization, X-ray characterization of crystals, data collection, processing and quality analysis of data, structure determination, molecular modeling, molecular docking and structure visualization and analysis. The facility is dedicated to X-ray diffraction and crystallographic studies and utilizes state of the art technology to understand the structural basis for biological function and dynamics. Transglutaminases (TGase; protein-glutamine: amine (gamma) glutamyl-transferase) are a diverse family of Ca+2 dependent enzymes with distinct genes, structures and biological functions. They are responsible for blood clotting, apoptosis, seminal fluid coagulation, extracellular matrix, bone formation, and barrier formation in stratified squamous epithelia. To date, six different TGases are expressed in epithelia and are thought to be involved in the assembly of the cell envelope (CE). At least two different TGase enzymes are known to be required for effective barrier formation and include: the TGase 1 enzyme, which is usually membrane bound, and the TGase 3 enzyme, which is cytosolic. It has been assumed that among all TGases, only TGase 2 binds GTP, and hydrolyzes it to GDP. The GTP bound TGase 2 appears to activate phopholipase C by lowering the calcium requirement of phopholipase C for substrate hydrolysis, and is therefore a crucial element for signal transduction. GTP binding by TGase 2 inhibits calcium binding resulting in the inhibition of TGase transamidation activity, whereas calcium binding inhibits GTP binding and activates the enzyme. Despite the availability of the TGase 2 structure bound to GDP, it remains unclear how calcium and GTP binding are able to reciprocally regulate the activity of the enzyme. The TGase 2 structure did not indicate the presence of bound divalent cations owing to its inactive state. We presented an extensive set of biochemical and crystallographic data showing how the TGase 3 enzyme interacts with GTP and with Ca2+/Mg2+. These data indicate for the first time that GTP binding is not restricted to the TGase 2 enzyme. Moreover, we showed that TGase 3 is able to hydrolyze GTP to GDP effectively. The crystal structures of TGase 3 in the presence of the non-hydrolyzable GTP analog GTPgammaS at 2.1? and also GDP at 1.9? showed three regions of switched conformations in comparison to our previous TGase 3 structure in the absence of guanine nucleotide. GTP binding is coordinated with a substitution of one of the three Ca2+ binding sites, and other subtle changes that cause the closing of a central channel leading to the enzyme's active site. Hydrolysis of GTP to GDP appears to impart a two-state conformation representing both the GTP bound enzyme (the GTPgammaS inactive state with Mg2+ion at site 3/closed channel) and the non-nucleotide bound (the active state with Ca2+ion at site 3/open channel) of the enzyme. In addition we reported the crystal structure determined at 2.0 ? resolution of TGase 3 in complex with GMP in order to elucidate the structural features required for nucleotide recognition. Binding affinities for various nucleotides were found by fluorescence displacement to be as follows: GTPgammaS (0.4 microM), GTP (0.6 microM), GDP (1.0 microM), GMP (0.4 microM), and ATP (28.0 microM). Furthermore, we found that GMP binds as a reversible, noncompetitive inhibitor of TGase 3 transamidation activity, similar to GTPgammaS and GDP. A genetic algorithm similarity program (GASP) approach (virtual ligand screening) identified three compounds from the Lead Quest TM database (Tripos Assoc. Inc) based on superimposition of GTPgammaS, GDP, and GMP guanine nucleotides from our crystal struuctures to generate the minimum align flexible fragment. These three were nucleotide analogs without a phosphate group, containing the minimal binding motif for TGase 3 that includes a nucleoside recognition groove. Binding affinities were measured as follows: TP349915 (Kd=4.1 microM), TP395289 (Kd=38.5 microM), TP394305 (Kd=1.0 mM). Remarkably, these compounds, do not inhibit, but instead activate TGase 3 transamidation by about 10-fold. These results suggest that the nucleotide-binding pocket in TGase 3 may be exploited to either enhance or inhibit the enzymatic activity as required for different therapeutic approaches. Results: These structures, backed with extensive biochemical studies, are providing new insights as to how access to the enzyme's active site may be gated through the coordinated changes in cellular calcium and magnesium concentrations and GTP/GDP. Calcium-activated TGase 3 can bind, hydrolyze, and is inhibited by GTP, despite lacking structural homology with other GTP binding proteins. A structure based sequence homology among the TGase enzyme family shows that these essential structural features are shared among other members of the TGase family. ClpXP is a bipartite chaperone/protease machine that catalyzes ATP-dependent protein unfolding and degradation in bacteria and in subcellular compartments of eukaryotes. It is an important global regulator in bacterial cells, where it targets specific short-lived proteins for degradation and plays essential roles in developmental changes, survival under starvation conditions, replication of phage and plasmids, and in various other regulatory. It also acts in protein quality control pathwa. ClpXP is present in the chloroplasts of plants and photosynthetic bacteria, where it has essential functions and is required for cell viability. In humans, hClpX is encoded on chromosome 15q22.2 and hClpP is encoded on chromosome 19p13.3. Both proteins are targeted to mitochondria, but the specific functions of ClpXP in human mitochondria have not been defined. ClpP is a self-compartmentalized protease; two rings of seven subunits enclose a hollow chamber containing the proteolytic active sites accessible only through axial channels in each ring. Substrates are presented to ClpP by ClpX or by the related chaperone, ClpA, each of which recognize specific proteins and target them for degradation. ClpX and ClpA are members of the Clp/Hsp100 family of molecular chaperones and belong to the AAA+ super-family of ATPases associated with various cellular activities, a diverse class of chaperones that disassemble and otherwise modify macromolecular complexes. ClpX and ClpA form stable hexameric rings in the presence of ATP, and the hexamers co-axially stack on each end of ClpP forming barrel-like holoenzyme complexes. Results: We have determined a 2.1? crystal structure for human mitochondrial ClpP (hClpP), the proteolytic component of the ATP-dependent ClpXP protease. HClpP has a structure similar to that of the bacterial enzyme, with the proteolytic active sites sequestered within an aqueous chamber formed by face-to-face assembly of the two heptameric rings. We propose that the N-terminal peptide of ClpP is a structural component of the substrate translocation channel and may play an important functional role as well.